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Biological roles of cellular glyceraldehyde-3-phosphate dehydrogenase in the hepatitis C virus life cycle

University of British Columbia

The hijacking and manipulation of host cell biosynthetic pathways by human viruses are shared molecular events that are essential for the viral life cycle. Because of increasing evidence of the importance of human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in host secretory pathway functions, I hypothesized that this multifunctional enzyme could contribute to life cycles of two Flaviviridae members, hepatitis C virus (HCV) and dengue virus (DENV). The first aim of this project was to investigate whether GAPDH is a host factor that regulates the life cycle steps of HCV in human hepatoma Huh-7.5.1 cells. I used short interfering RNA (siRNA)-mediated silencing of GAPDH both pre- and post-HCV infection in Huh-7.5.1 cells to demonstrate that reducing GAPDH protein abundance inhibits primary HCV infection and production and/or release of infectious HCV virus particles. Exogenous expression of V5-tagged human GAPDH, pre- and post-infection, increases the viral infectivity of HCV-infected Huh-7.5.1 cell supernatants, suggesting a predominant role of GAPDH during the post-replication steps of the HCV life cycle. Finally, siRNA-mediated GAPDH suppression in human Huh-7.5.1 cells also significantly inhibited primary DENV-2 infection. In the second aim, I further investigated the diverse functions of GAPDH in human hepatoma cells by performing differential expression profiling of total cellular proteins by quantitative proteomics in two GAPDH knockdown Huh-7-derived cell lines (67D2 and 67b3) and the parental Huh-7 cell line. First, I successfully established GAPDH knockdown Huh-7-derived cell lines using short hairpin RNA (shRNA) lentivirus particles. Second, I demonstrated that the stable shRNA-mediated GAPDH silencing in Huh-7 cells inhibits primary HCV infection and the production of infectious HCV virus particles. Using a quantitative proteomics strategy based on triplex dimethyl labeling and nano-liquid chromatography-tandem mass spectrometry, I determined the cellular proteins deregulated in 67D2 and 67B3 cells. Bio-informatic analysis of the differentially expressed proteins revealed a robust compensatory effect in molecular functions associated with enzymatic activities and “acting binding” in response to the silencing of GAPDH in 67D2 and 67D3 cells.

Text

2015-08-04

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/54275

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/